Fetal Alcohol Spectrum Disorder (FASD) describes a wide array of ethanol-induced developmental defects, including craniofacial dysmorphology, such as lower jaw hypoplasia. It affects approximately 1 in 100 children born in the United States each year. Although fetal exposure to alcohol causes FASD, virtually nothing of the genetics behind these ethanol-induced craniofacial defects is understood. The majority of the craniofacial skeleton, including the lower jaw, is generated by cranial neural crest cells. Complex interactions between neural crest cells and epithelial tissues are important for craniofacial morphogenesis. These interactions are orchestrated by, among others, the Bone Morphogenic Protein (Bmp) signaling pathway. Preliminary data using zebrafish demonstrate that members of the Bmp signaling pathway are ethanol-sensitive loci. While bmp2b homozygous mutant embryos die before craniofacial development initiates, bmp2b heterozygous embryos and bmp4 homozygous mutant embryos (together termed Bmp-pathway mutants) undergo normal craniofacial development. However, Bmp-pathway mutants display ethanol-induced defects to Meckel's cartilage the lower jaw. Mutation of the sphingosine receptor, s1pr2, causes strikingly similar defects to Meckel's cartilage due to disrupted morphogenesis of the anterior endoderm and subsequent defects to the oral ectoderm. The findings suggest the hypothesis that ethanol disrupts anterior endoderm morphogenesis in Bmp-pathway mutants resulting in defects in the oral ectoderm and subsequent craniofacial development. In Specific Aim 1 of this proposal, 1) how ethanol perturbs cell behaviors during endodermal morphogenesis in Bmp-pathway mutants will be visualized and 2) the interaction between the endoderm and oral ectoderm will be directly visualized through generation of a double transgenic line that labels the endoderm and the oral ectoderm. In Specific Aim 2, the mechanism of Bmp-pathway signaling in patterning the lower jaw will be determined through transplantation experiments and in situ marker analyses. In Specific Aim 3, an ethanol- sensitive mutant recovered from a forward genetic screen will be mapped and the overall ethanol sensitivity characterized. Overall, this application will provide greater insight into the gene-ethanol interactions that lead to the variability of craniofacial defects in FASD. In addition, because of the conservation of gene function between zebrafish and humans, this work will directly translate to studies of candidate genes in human populations and allow better for diagnosis and treatment of FASD.